Estimation of Divergence Times for Major Lineages of Primate Species

Galina V. Glazko and Masatoshi Nei

Institute of Molecular Evolutionary Genetics and Department of Biology, Pennsylvania State University,
University Park, Pennsylvania

Although the phylogenetic relationships of major lineages of primate species are relatively well established, the times of
divergence of these lineages as estimated by molecular data are still controversial. This controversy has been generated in
part because different authors have used different types of molecular data, different statistical methods, and different
calibration points. We have therefore examined the effects of these factors on the estimates of divergence times and
reached the following conclusions: (1) It is advisable to concatenate many gene sequences and use a multigene gamma
distance for estimating divergence times rather than using the individual gene approach. (2) When sequence data from
many nuclear genes are available, protein sequences appear to give more robust estimates than DNA sequences. (3)
Nuclear proteins are generally more suitable than mitochondrial proteins for time estimation. (4) It is important first to
construct a phylogenetic tree for a group of species using some outgroups and then estimate the branch lengths. (5) It
appears to be better to use a few reliable calibration points rather than many unreliable ones. Considering all these factors
and using two calibration points, we estimated that the human lineage diverged from the chimpanzee, gorilla, orangutan,
Old World monkey, and New World monkey lineages approximately 6 MYA (with a range of 57), 7 MYA (range,
68), 13 MYA (range, 1215), 23 MYA (range, 2125), and 33 MYA (range 3236).

Introduction

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In recent years a large number of authors have
investigated the evolutionary relationships of primate
species, using both molecular and paleontological data,
and we now have a rough picture of the phylogenetic
relationships of the major lineages of primate species
(e.g., Goodman et al. 1998). However, the times of
divergence of these lineages are still controversial (e.g.,
Horai et al. 1995; Takahata and Satta 1997; Arnason,
Gullberg, and Janke 1998; Arnason et al. 2000; Cao et
al. 2000; Chen and Li 2001). For example, the estimate
of the time of divergence between humans and
chimpanzees varies from 3.6 MYA (Easteal and Herbert
1997) to 13 MYA (Arnason, Gullberg, and Janke 1998).
This controversy has occurred because different authors
have used different types of molecular data (e.g., nuclear
genes, mitochondrial genes, and noncoding DNA
regions), different statistical methods, and different
calibration points.
Estimation of divergence time is generally more
difficult than reconstruction of a phylogenetic tree, be-
cause, strictly speaking, no gene would evolve at a con-
stant rate. For this reason, recent authors have used many
independently evolving genes to estimate divergence
times in the hope of reducing the effect of rate varia-
tion (e.g., Doolittle et al. 1996; Wray, Levinton, and Shapiro
1996; Kumar and Hedges 1998). The traditional method
of using information from many different genes is to
compute an estimate of divergence time between two
species or two groups of species for each gene and then take
the average of all the estimates (individual gene [IG] or
individual protein [IP] approach). Nei, Xu, and Glazko
(2001) showed that this method tends to give biased
estimates of divergence times, particularly overestimates
when the calibration date is smaller than the time to be
Key words: divergence times, concatenated distance, primate
species, calibration points.
E-mail: nxm2@psu.edu.
Mol. Biol. Evol. 20(3):424434. 2003
DOI: 10.1093/molbev/msg050
2003 by the Society for Molecular Biology and Evolution. ISSN: 0737-4038
estimated. They then proposed the concatenated distance
method, in which some sorts of concatenated distances for
all genes are first computed and the divergence time is then
estimated from the distances for all pairs of species. In
particular, they suggested the use of a gamma distance for
concatenated sequences (CS) for all genes (multigene or
multiprotein gamma distance).
It has been customary to use protein sequences rather
than DNA sequences for time estimation, because the
former are generally more conserved than the latter and
can be handled by simpler mathematical models (e.g.,
Doolittle et al. 1996; Kumar and Hedges 1998; Nei and
Kumar 2000, chapter 10). Some authors have argued that
because noncoding regions of DNA sequences are not
direct targets of natural selection, they should give more
reliable estimates (Goodman et al. 1998; Chen and Li
2001). However, noncoding regions are subject to in-
sertion and deletion more often than coding regions, and
therefore they may not necessarily give reliable estimates.
Nevertheless, for estimating relatively short evolutionary
times, as in the present case, DNA sequences both for
coding and noncoding regions may be more informative
than protein sequences. Because of abundant availability,
mitochondrial (mt) DNA have also been used extensively
for time estimation in the past (e.g., Horai et al. 1995;
Arnason et al. 1996, 1998, 2000). However, the estimates
obtained from mt genes are controversial because the
evolutionary rate of mt genes apparently varies rather
extensively among different groups of mammals (Gissi
et al. 2000). We have therefore decided to compare
estimates of divergence times obtainable from nuclear
protein-coding genes, noncoding DNA sequences, and mt
genes.
One of the important factors that determine the
accuracy of estimates of divergence times is reliability of
the calibration point used for producing the time scale of
the phylogenetic tree constructed. In this study we use the
times of divergence between humans and orangutans
(about 13 MYA) and between primates and artiodactyls
(90 MYA) as calibration points. We are interested in

424

FIG. 1.(A) Neighbor-Joining tree for hominoid 4 species and
artiodactyls constructed by using multiprotein gamma distance (dMG)
with a 5 0.47 for 29 protein sequences. Two rodent species were used as
outgroups. (B) Linearized tree of the above tree. The timescale does not
apply to rodents, because these species are outgroups.
finding whether these two calibration points give similar
time estimates for other branch points in the tree.
Materials and Methods
Species and Genes Used
Our intention in this study was to use as many genes
as possible for a group of species under consideration.
However, the number of gene sequences available for
primate species is quite limited except for a few primate
species. Furthermore, to estimate divergence times, we
needed species that would provide reasonably good
calibration points. One of the calibration points we used,
as already mentioned, is the time of divergence between
the orangutan and the human lineages (13 MYA). This
fossil dating based on Sivapithecus was once questioned
(Pilbeam et al. 1990), but recent statistical analyses of
cranial and postcranial characters (Begun, Ward, and Rose
1997; Ward 1997) suggest that the Sivapithecus-Pongo
(orangutan) clade remains the strongest phylogenetic
hypothesis (Ward 1997). We therefore decided to use
this calibration point, and for this reason inclusion of
orangutan genes was essential. Another fossil-based
calibration point we used was the time of divergence
between archaic fossil ungulates (clade Ungulatomorpha)
and primates. The fossils indicate that Ungulatomorpha
appeared about 8590 MYA (Archibald 1996; Archibald,
Averianev, and Ekdale 2001). Because fossils generally
give a minimum estimate of splitting time, we assumed
that primates and artiodactyls diverged 90 MYA and used
primarily cattle (Bos taurus) genes for artiodactyls.
For estimating the divergence times for a group of
species, we have to have outgroups to determine the root
of the tree for the group (fig. 1). For this purpose, we used
mice and rats (see Results for the justification of using
Divergence Times of Major Primate Lineages 425
rodents as outgroups). However, the number of genes
available varied considerably with species. We therefore
conducted separate analyses for the following four species
groups.
Species Group 1
For this group, we could use 29 shared nuclear genes.
The primate species used were humans (Homo sapiens),
chimpanzees (Pan troglodytes), gorillas (Gorilla gorilla),
and orangutans (Pongo pygmaeus), and their topological
relationships are given in figure 1A. Artiodactyls (primar-
ily cattle genes) were used for calibrating evolutionary
times, whereas mice (Mus musculus) and rats (Rattus
norvegicus) were used for determining the root of the tree
for the remaining species. The names of the genes used are
listed in table S1 of the online Supplementary Material and
the Web site http://www.bio.psu.edu/people/faculty/nei/
lab/databases.htm. The smallest number of genes available
from GenBank was for orangutans, and we compiled all
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genes that are orthologous to the orangutan genes. To
avoid paralogous genes, we constructed a Neighbor-
Joining (NJ) tree (Saitou and Nei 1987) for each gene
using uncorrected p distance (Nei and Kumar 2000, p. 18)
and eliminated all the genes that produced the incorrect
topology except for humans, chimpanzees, and gorillas, or
the genes that showed zero distances for all pairs of
humans and African ape species. Apparently because
humans, chimpanzees, and gorillas diverged in a short
period of evolutionary time and the ancestral populations
were polymorphic, different genes are known to show
different topologies for the three species (Saitou and
Nei 1986; Satta, Klein, and Takahata 2000; Chen and Li
2001; Klein and Takahata 2002; OhUigin et al. 2002).
Therefore, we cannot eliminate paralogous genes for these
species by comparing the gene tree with the species tree. In
this case we used the most plausible orthologous genes,
although there were only a few such cases. The final
number of genes chosen in this way was 29 protein-coding
genes with a total of 6,966 codons.
Species Group 2
For estimating the divergence times for Old World
(OW) monkeys (mostly macaque [Macaca mulatta] genes)
and New World (NW) monkeys (mostly marmoset
[Callithrix jacchus] genes), we could obtain only 13
nuclear genes with 2,425 codons. Therefore, we conducted
a separate analysis for the nine species listed in figure 2A.
The genes used in this study were a subset of the genes
used for species group 1 (see online Supplementary
Material).
Species Group 3
Species group 3 was chosen primarily for studying
time estimates obtainable from noncoding regions of
DNA sequences. In this group we estimated the times of
divergence of the human lineage from the chimpanzee,
gorilla, orangutan, and OW monkey (macaque) lineages
and used NW monkeys (marmoset) as the outgroup
(fig. 3). The noncoding DNA regions used were the
flanking and intergenic regions of the e and the c1-c2
426 Glazko and Nei
FIG. 2.(A) Neighbor-Joining tree for simian primates and
artiodactyls constructed by using multiprotein gamma distance with a 5
0.61 for 13 protein sequences. (B) Linearized tree of the above tree. The
time scale does not apply to rodents, because these species are outgroups.
globin genes (9,818 bp) (http://cmmg.biosci.wayne.edu/
lgross/). The noncoding sequences of the globin gene
regions are available even for loris, lemurs, and others
(Goodman et al. 1998). However, because the comparison
of sequences from distantly related species showed many
deletions and insertions, we did not include these species in
this study.
Species Group 4
For some unknown reasons, the evolutionary rate of
mt genes varies considerably from species to species (Gissi
et al. 2000), and therefore these genes may not give
reliable estimates of divergence times. However, a large
number of authors have used these genes for studying
primate evolution. We have therefore examined time
estimates obtainable from mt genes. In this study we used
humans, chimpanzees, gorillas, orangutans, gibbons,
baboons (Papio hamadryas; OW monkeys), capuchins
(Cebus albifrons; NW monkeys), slow loris (Nycticebus
coucang; strepsirhines), artiodactyls (Bos taurus), and two
rodent species (fig. 4A). Each of these species has 13
coding genes, and we used all the genes, regardless of the
FIG. 3.Neighbor-Joining tree for simian primates constructed by
using Kimura distance for the noncoding regions of the e and the c1-c2
genes.
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FIG. 4.(A) Neighbor-Joining tree for simian and prosimian
primates and non-primate mammalian species obtained by multiprotein
gamma distances (dMG) for 13 mitochondrial proteins (3,752 amino acids)
with a 5 0.51. *Species that evolved significantly faster the average.
Species that evolved significantly more slowly. Two rodent species
were used as outgroups. (B) Enforced linearized tree of tree A.
transcription direction. Some authors (e.g., Arnason et al.
1996, 2000) excluded genes NAD6 and COX2 because of
the difference in transcription direction or a higher rate of
evolution in some species groups. However, our pre-
liminary study showed that inclusion or exclusion of these
genes has little effect on time estimates.
Statistical Methods
As already mentioned, we are primarily interested in
the CS approach in this study. When protein sequences
were used, we first constructed a NJ tree using MEGA2
(Kumar et al. 2001) with multiprotein gamma distance
(dMG), which is a Poisson-correction (PC) gamma distance
obtained for the concatenated amino acid sequences for all
the proteins used. The gamma parameter a was estimated
by Gu and Zhangs (1997) method. In practice, however,
the a value obtained by this method is sometimes too small
for the purpose of time estimation (Nei, Xu, and Glazko
2001), and the classical Dayhoff distance, which can be
obtained by a PC gamma distance with a 5 2.25 (Nei and
Kumar 2000, p. 23), often gives reasonable time estimates
when the divergence time considered is relatively short.
Actually, it is known that even PC distance with a 5
gives reasonable time estimates when conserved proteins
such as cytochrome c and hemoglobins are used (Dick-
erson 1971). Because the proteins we used were quite
conserved, we used Dayhoff and PC distances as well.
When we analyzed DNA sequences, the Jukes-Cantor,
Kimura, and Kimura gamma distances (Nei and Kumar

FIG. 5.Cauchy distribution compared with the normal distribution.
p(^t) refers to the probability density of ^t.
2000, chapter 3) were used. Although the phylogenetic
tree of the primates species used here is reasonably well
established (Goodman et al. 1998), we also constructed
maximum parsimony (MP) trees using the branch-and-
bound algorithm of MEGA2 and maximum likelihood
(ML) trees using the Poisson model of PROTML (Adachi
and Hasegawa 1996) for protein data and PAUP*
(Swofford 1998) for DNA data. The topologies of these
trees were always the same as those of NJ trees.
Once the topology of the species was determined, the
branch lengths of the tree were estimated by the least
squares method. We then used Takezaki, Rzhetsky, and
Neis (1995) two-cluster and branch-length tests to
examine the molecular clock hypothesis (computer pro-
gram LINTREE; see http://mep.bio.psu.edu). Only when
these tests were significant at the 1% level did we consider
the deviation biologically meaningful, because reasonably
good time estimates are known to be obtained even if the
deviation is considerably large (Nei and Kumar 2000,
chapter 10). When the molecular clock hypothesis was
acceptable, we constructed a linearized tree to estimate the
times of species divergence. When it was rejected, we used
the stem-lineage method proposed by Nei, Xu, and Glazko
(2001) and Nei and Glazko (2002).
As mentioned earlier, the traditional method of time
estimation is first to construct a linearized tree for each
gene with a gamma distance and estimate the divergence
times for this tree. Let us consider the linearized tree in
figure 1B and assume that the divergence time (T1) be-
tween primates and artiodactyls is known (90 MYA). The
divergence time (t) between humans and chimpanzees can
then be estimated by
^t a=bT1 ;
where a and b are branch length estimates for the human
and artiodactyl lineages, respectively, in figure 1B. If ^t is
computed for all genes examined, t is estimated by their
average (
t ). That is,
X
k

t ^ti =k;
i1
where i stands for the ith gene and k is the number of genes
used. If a and b are normally distributed and k is large, the
Divergence Times of Major Primate Lineages 427
distribution of
t is known to be given by the following
Cauchy distribution:

1
p
t p
1 1
t 2 3
(Johnson and Kotz 1970, p. 154). This has a wider
distribution than the normal distribution, and the theoret-
ical variance is infinite (fig. 5). One way to avoid the
problem of large variances is to eliminate so-called outliers
(say 5% of uppermost and 5% lowermost ^ti values)
(Kumar and Hedges 1998). In this approach, however, it is
unclear how ^t is affected by the elimination, and some
subjective judgment may enter into the computation. Nei,
Xu, and Glazko (2001) and Nei and Glazko (2002) also
showed that
t tends to be an overestimate when the
divergence time to be estimated is older than the
calibration point.
By contrast, if we use a multigene gamma distance
for concatenated sequences of many genes and estimate t1
by equation (1) using a and b obtained from multigene
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distances, the bias inherent in equation (2) virtually
disappears. The standard errors of ^t1 is computed by the
bootstrap test using genes (rather than amino acids or
nucleotides) as the units of resampling. This computation
can be made by our computer program TIMER (http://
mep.bio.psu.edu).
Results
Species Group 1
We first analyzed protein sequences following Nei,
Xu, and Glazko (2001). The phylogenetic tree presented in
figure 1A is an NJ tree obtained by using multiprotein
gamma distance (dMG) with a 5 0.47. The topology of this
tree is well supported by the bootstrap test. It was also
supported by MP and ML analyses. The topology of the
primate portion of the tree is identical with the generally
accepted molecular topology (Goodman et al. 1998).
However, the remaining portion of the tree is somewhat
controversial. There is no fossil record that resolves the
evolutionary relationships of primates, artiodactyls, and
rodents (Bromham, Phillips, and Penny 1999). Most
molecular studies in the past have supported a close
relationship between primates and artiodactyls rather than
between primates and rodents or between artiodactyls and
rodents (e.g., Li et al. 1990; Cao et al. 1998; Arnason et al.
2000; Reyes, Pesole, and Saccone 2000). Recently,
however, Murphy et al. (2001) constructed a phylogenetic
tree of about 42 placental mammalian species using 19
nuclear and 3 mitochondrial genes and suggested that
1 primates and rodents are phylogenetically closer to each
other than to artiodactyls. However, our phylogenetic
analysis of 71 nuclear proteins with 24,952 amino acids
for humans, artiodactyls, rodents, chicken, and Xenopus
has generated the traditional molecular topology with
a high level of statistical support (Nei and Glazko 2002).
We have therefore decided to use the phylogenetic tree
2 given in figure 1 for our study of divergence times of
hominoid species. For estimating divergence times for
primate species, however, the topological relationships
among primates, artiodactyls, and rodents do not matter
428 Glazko and Nei

Table 1
Estimates (6 Standard Errors) of Divergence Times of the Human Lineage from Other
Primate Species and Artiodactyls Obtained by Using Different Approaches (29 Nuclear
Proteins)
Calibration Point Chimp Gorilla Orangutan Artiodactyl
Concatenated sequence (CS) approach
Multiprotein gamma (dMG with a 5 0.47)
T1 5 90 MYA 5.5 6 0.6 6.2 6 0.7 12.5 6 0.7 90
T2 5 13 MYA 5.7 6 0.5 6.5 6 0.7 13 93.3 6 5.5
Dayhoff distance (dMG with a 5 2.25)
T1 5 90 MYA 6.3 6 0.8 7.2 6 0.6 14.3 6 0.7 90
T2 5 13 MYA 5.8 6 0.6 6.6 6 0.5 13 81.5 6 4.6
PC distance (dMG with a 5 )
T1 5 90 MYA 6.6 6 0.8 7.5 6 0.7 14.8 6 0.8 90
T2 5 13 MYA 5.8 6 0.6 6.6 6 0.7 13 79.0 6 4.4
Individual gene (IG) approach
Gamma distance (
a 5 0.80)
T1 5 90 MYA 4.3 6 0.8 8.2 6 2.4 12.8 6 1.6 90
T2 5 13 MYA 5.0 6 0.8 7.2 6 1.1 13 157.1 6 30.3
Dayhoff distance (a 5 2.25)
T1 5 90 MYA 4.8 6 0.9 8.9 6 2.5 14.3 6 1.7 90
T2 5 13 MYA 5.0 6 0.8 7.3 6 1.1 13 141.7 6 28.0

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PC distance (a 5 )
T1 5 90 MYA 5.0 6 0.9 9.1 6 2.5 14.7 6 1.7 90
T2 5 13 MYA 5.1 6 0.8 7.3 6 1.1 13 134.8 6 27.1

very much, because the linearized tree is constructed
without rodent species.
To estimate the divergence times, we first tested the
molecular clock hypothesis by using Takezaki, Rzhetsky,
and Neis (1995) method. The results of this test showed
that the deviation from the clock hypothesis is not
statistically significant at the 1% level. We therefore
constructed the linearized tree (fig. 1B). The evolutionary
time scale given for this tree was obtained by using the
divergence time between primates and artiodactyls (T1 5
90 MYA) as the calibration point and a rate of amino acid
substitution of 1.2 3 1029 per year per lineage. Estimates
of the divergence times for humans and ape species are
presented in table 1. The estimates obtained by using the
human/orangutan divergence (T2 5 13 MYA) as the
calibration point are also presented in table 1. It is
interesting to note that the two sets of estimates are close to
each other and that the estimate of divergence time
between humans and chimpanzees (about 5.55.7 MYA)
is close to the ages of the recently discovered oldest
hominid fossils (5.47.0 MYA; Aiello and Collard 2001;
Haile-Selassie 2001; Brunet et al. 2002). When the second
calibration point (T2 5 13 MYA) is used, we obtained 93
MYA for the divergence time between primates and
artiodactyls. This estimate is close to T1 5 90 MYA. Table
1 also includes estimates obtained by using Dayhoff and
PC distances. These distances give reasonably good time
estimates for humans and apes if we assume that the lower
limit of the human/chimpanzee divergence time is about
5.4 MYA and the upper limit of the primate/artiodactyl
divergence time is about 90 MYA. These results show that
when a ranges from 0.47 to the time estimates remain
nearly the same for these relatively closely related species.
(dMG with a 5 0.47 is better for estimating the primate/
artiodactyls divergence time.)
Table 1 includes the time estimates obtained by the
IG approach with gamma distances. In this case the time
estimate for the human/chimpanzee divergence is appar-
ently too small when T1 5 90 MYA was used as the
calibration point, and the estimate for the primate/
artiodactyl divergence is too large when T2 5 13 MYA
was used (75% overestimate of the calibration date). This
type of underestimation and overestimation occurs even
when Dayhoff distance or PC distance is used.
In the present data set, the extent of protein
divergence among the human and ape species was rather
small, so we suspected that DNA sequences might give
more reliable results. We therefore estimated the di-
vergence times using the concatenated DNA sequences for
the species in figure 1A, for which the DNA sequences of
24 genes were available. (No DNA sequences were
available for five genes for some of the species used.) In
this study we first used the Kimura gamma distance for the
entire sequences (18,272 bp), and then for the sequences of
first and second codon positions only. The results obtained
are presented in table 2. When we used all three codon
positions of DNA sequences and T1 5 90 MYA as the
calibration point, the estimates of divergence times among
hominoid species appeared to be too low, but the primate/
artiodactyl divergence time was apparently overestimated
when T2 5 13 MYA was used as the calibration point.
This tendency did not change, even when we used the
sequence data for first and second codon positions and
Kimura gamma distance. These results suggest that protein
data generally give more reliable estimates than DNA data
even for closely related species.
Interestingly, however, DNA data gave reasonably
good estimates of the human/orangutan and the primate/
artiodactyl divergence times when Kimura distance rather
than Kimura gamma distance was used for first and second
codon position data. This finding might suggest that the
parameter for the gamma distance a is underestimated for
closely related sequences and that Kimura distance for first
and second codon positions is a good option in this case.
 Divergence Times of Major Primate Lineages 429

Table 2
Estimates of Divergence Times (6 Standard Errors) of the Human Lineage from Other
Primate Species and Artiodactyls Obtained by Using Nucleotide Sequences of 24 Genes
Calibration point Chimp Gorilla Orangutan Artiodactyl
All three codon positions used
Kimura gamma distance (a 5 0.73)
T1 5 90 MYA 4.1 6 0.2 4.7 6 0.3 9.1 6 0.3 90
T2 5 13 MYA 5.8 6 0.2 6.7 6 0.3 13 128.76 5.3
Kimura distance
T1 5 90 MYA 4.8 6 0.4 5.4 6 0.4 10.5 6 0.4 90
T2 5 13 MYA 6.0 6 0.3 6.7 6 0.4 13 111.9 6 5.3
1st and 2nd codon positions used
Kimura gamma distance (a 5 0.40)
T1 5 90 MYA 5.0 6 0.2 5.6 6 0.2 10.3 6 0.4 90
T2 5 13 MYA 6.3 6 0.2 7.1 6 0.2 13 113.7 6 5.1
Kimura distance
T1 5 90 MYA 6.1 6 0.3 6.7 6 0.3 11.6 6 0.4 90
T2 5 13 MYA 6.6 6 0.2 7.3 6 0.2 13 97.8 6 4.1

However, this is somewhat illogical, because there must be respectively. These estimates are close to the rough

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rate heterogeneity for concatenated sequences. Therefore,
we are not sure whether we should give much weight to
this result.
Species Group 2
The time estimates obtained by multiprotein gamma
distance (a 5 0.61) with T1 5 90 MYA and T2 5 13
MYA for this species group are presented in table 3. They
are similar to each other and are rather close to the
estimates in table 1, whenever comparable estimates are
available. The estimate for the divergence between
humans and orangutans (12.6 MYA) is also close to the
paleontological estimate (13 MYA). Similarly, the esti-
mate for the divergence time between primates and
artiodactyls (92.8 MYA) is close to the paleontological
data (90 MYA). Actually, these statements hold true even
with Dayhoff and PC distances, although the primate/
artiodactyls divergence time tends to be underestimated
when T2 5 13 MYA is used. The average estimates of the
time of divergence between humans and OW monkeys and
NW monkeys are approximately 23 MYA and 33 MYA,
estimates obtained by Goodman et al. (1998).
By contrast, the IG approach again gives unduly low
estimates for the human/chimp divergence but gives an
unduly high estimate for the primates/artiodactyl diver-
gence, as expected theoretically (Nei, Xu, and Glazko
2001; Nei and Glazko 2002). When T2 5 13 MYA is used,
the estimates of the time of divergence of humans from
OW monkeys and NW monkeys are also considerably
higher than those obtained by the CS approach.
We also used all three codon position data and first
and second codon positions of DNA sequences to estimate
divergence times. In this case we could use only nine
genes. The results were quite similar to those presented in
table 2. That is, when Kimura distance or Kimura gamma
distance for all three codon positions was used, the
calibration point of T1 5 90 MYA gave too low estimates
for the human/chimp and the human/gorilla divergence,
whereas T2 5 13 MYA gave a too high estimate of the
primate/artiodactyl divergence (table S1 of the online
Supplementary Material). By contrast, when Kimura
distance was used for first and second codon position
data, both T1 5 90 MYA and T2 5 13 MYA gave
reasonable estimates. However, the estimates obtained

Table 3
Estimates of Divergence Times (6 Standard Errors) of the Human Lineage from Other Primate Species and
Artiodactyls (13 Nuclear Proteins)
Calibration Point Chimp Gorilla Orangutan Old World Monkeys New World Monkeys Artiodactyl
Concatenated sequence (CS) approach
Multiprotein gamma (dMG with a 5 0.61)
T1 5 90 MYA 6.1 6 1.0 6.4 6 0.8 12.6 6 1.2 21.6 6 1.6 31.9 6 2.0 90
T2 5 13 MYA 6.3 6 0.9 6.6 6 1.0 13 22.3 6 2.0 33.0 6 3.2 92.8 6 8.9
Dayhoff distance (dMG with a 5 2.25)
T1 5 90 MYA 7.1 6 1.1 7.5 6 1.0 14.4 6 1.3 24.3 6 1.6 35.2 6 2.0 90
T2 5 13 MYA 6.4 6 1.0 6.7 6 0.8 13 21.9 6 1.9 31.8 6 3.0 81.2 6 7.4
PC distance (a 5 )
T1 5 90 MYA 7.4 6 1.1 7.8 6 1.0 15.0 6 1.3 25.2 6 1.7 36.3 6 2.0 90
T2 5 13 MYA 6.4 6 1.0 6.7 6 0.9 13 21.8 6 1.9 31.4 6 3.0 77.9 6 7.0
Individual gene (IG) approach (
a 5 1.05)
T1 5 90 MYA 3.2 6 1.2 5.9 6 2.5 10.0 6 2.1 21.7 6 3.7 30.8 6 3.5 90
T2 5 13 MYA 4.7 6 1.3 7.8 6 1.7 13 38.1 6 7.3 64.9 6 12.4 229.7 6 60.7
430 Glazko and Nei

were again similar to those obtained from multiprotein Table 4

gamma distance with a 5 0.61. Estimates (6 Standard Errors) of Divergence Times
(MYA) of the Human Lineage from Other Primate
Species Obtained from Noncoding DNA Sequences of the e
Species Group 3 and g1-g2 Globin Gene Regions
The phylogenetic tree for the noncoding regions of the Chimp Gorilla Orangutan Old World Monkeys
e and the c1-c2 gene clusters is presented in figure 3. When Jukes-Cantor distance
the marmoset (NW monkey) was used as the outgroup, the 5.5 6 0.2 6.0 6 0.2 13 24.8 6 0.6
evolutionary change of hominoid and macaque sequences Kimura distance
5.5 6 0.2 6.0 6 0.2 13 24.8 6 0.5
did not deviate significantly from the molecular clock. We Kimura gamma distance (a 5 0.26)
therefore constructed a linearized tree for the hominoids and 5.2 6 0.2 5.7 6 0.3 13 26.8 6 0.7
OW monkeys and estimated the divergence times for these
NOTE.The total number of nucleotides used is 9,818. The human/orangutan
species using the human/orangutan divergence time as the divergence time was used as the calibration point.
calibration point (table 4). When Kimura gamma distance
with a 5 0.26 was used, the estimated times of divergence of
humans from chimpanzees and gorillas were slightly smaller Janke (1998) (13, 16, 30, 35, 52, 70, and 90 MYA,
than those obtained by multiprotein gamma distance (tables respectively). Arnason, Gullberg, and Janke (1998)
1 and 3), but the estimate time for OW monkeys was slightly actually used some kind of rate-adjustment method,
higher. When Kimura or Jukes-Cantor distance was used, because they were aware of the slow rate of evolution of

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the results hardly changed. However, this study is not very
informative, because we could not use the calibration point
of T1.
Species Group 4
Figure 4A shows the NJ tree for eight primate and
three nonprimate species obtained by 13 mtDNA-encoded
proteins with 3752 amino acids. Essentially the same tree
topology was obtained by MP and ML analyses. In this
case the molecular clock obviously does not work, and the
orangutan, baboon, and capuchin monkey sequences
evolved significantly faster than the average sequence
(1% level), whereas the slow loris and cattle sequences
evolved significantly slower.
Therefore, the linearization of the tree cannot be
justified unless we eliminate all deviant species. However,
if we eliminate the deviant species, we have no more
calibration points. So, despite this clear violation of the
molecular clock, we attempted to construct a linearized
tree using all species. We again used the primate/
artiodactyl divergence time (T1 5 90 MYA) and the
human/orangutan divergence times (T2 5 13 MYA) as the
calibration points (table 5). The results were unexpectedly
interesting, because when T1 5 90 MYA was used, the
estimates of times of divergence of the human lineage
from the chimpanzee, gorilla, orangutan, gibbon, baboon,
capuchin, and slow loris lineages were approximately 11,
15, 32, 33, 63, 90, and 90 MYA, and these estimates are
very similar to those obtained by Arnason, Gullberg, and
the artiodactyl sequence, but their rate adjustment was
probably insufficient, because we obtained essentially the
same results without any rate adjustment. The inadequacy
of the linearized tree method in this case is also clear from
the fact that the use of the calibration point T2 5 13 MYA
gives estimates very different from those obtained by using
T1 5 90 MYA.
Nei, Xu, and Glazko (2001) and Nei and Glazko
(2002) suggested that the stem-lineage method for
estimating divergence times may be used when the
evolutionary rate varies with exterior branch and the
number of amino acids or nucleotides used is very large. In
this method the stem lineage that keeps generating exterior
branches is assumed to evolve at a constant rate, and the
divergence times are estimated by using only the stem
lineage. As in figure 4A, denoting the lengths of the
exterior and interior branches of the primate stem lineage
by a, b, c, d, e, f, g, h, and i, we can assume that the sum of
stem branch lengths, S 5 (a 1 b)/2 1 c 1 d 1 e 1 f 1
g 1 h 1 i, corresponds to the primate/cattle divergence
time, i.e., T1 5 90 MYA, and that the divergence time for
a pair of species is proportional to the appropriate sum of
branch lengths in the stem lineage. For example, we can
estimate the divergence time between humans and
baboons by [(a 1 b)/2 1 c 1 d 1 f] T1/S. In the case
of dMG with a 5 0.51 we have a 5 0.022, b 5 0.024, c 5
0.007, d 5 0.026, e 5 0.010, f 5 0.031, g 5 0.059, h 5
0.075, i 5 0.006, and S 5 0.237. Therefore, we obtain
37.2 MYA. By contrast, if we use T2 5 13 MYA as the
calibration point, the human/baboon divergence time is

Table 5
Estimates (6 Standard Errors) of Divergence Times (MYA) of the Human Lineage from Other Primate Species and
Artiodactyls Obtained by Using Multiprotein Gamma Distances (13 Mitochondrial Proteins with a 5 0.51)
Chimp Gorilla Orangutan Gibbon Baboon Capuchin Slow Loris Artiodactyl
Time estimates (dMG) obtained by linearized tree method
4.4 6.0 13 13.5 25.7 37.5 37.5 37.5
10.8 14.6 31.7 32.9 62.5 90.0 90.0 90.0
Time estimates obtained by using stem-lineage method
5.3 6 0.6 6.9 6 0.8 13 15.3 6 1.2 22.5 6 1.9 36.1 6 5.8 53.4 6 8.3 54.6 6 8.5
8.7 6 1.3 11.4 6 1.8 21.4 6 3.4 25.2 6 3.0 37.2 6 4.6 59.5 6 2.6 87.9 6 2.3 90

estimated by [(a 1 b)/2 1 c 1 d 1 f ] T2/S, where T2 5 13
MYA and S 5 (a 1 b)/2 1 c 1 d 5 0.056. Therefore, we
have 22.5 MYA.
The estimates of divergence times obtained for other
species are presented in table 5. The time estimates
obtained by using T1 5 90 MYA are again much higher
than those in tables 1 and 3, but those obtained by using
T2 5 13 MYA are rather close to those in the latter tables
except for artiodactyls. Nevertheless, because the stem-
lineage method depends on an unproven assumption, we
had better not to give much weight to these estimates.
We conducted a similar statistical analysis for the
DNA sequences of 13 coding genes using first, second,
and third codon positions. The time estimates obtained
were even more divergent from those given in tables 1 and
2, and the estimates obtained under the assumptions of
T1 5 90 MYA and T2 5 13 MYA were even more
inconsistent than those for protein data (data not shown).
These results again suggest that mt DNA sequence data are
less suitable for time estimation than nuclear proteins.
Discussion
In this article we have presented several problems
related to the estimation of divergence times. First, we
have shown that the IG method of time estimation often
gives biased estimates of divergence times, as was shown
theoretically by Nei, Xu, and Glazko (2001) and Nei and
Glazko (2002). This method tends to give overestimates of
divergence times when the calibration point is younger
than the estimated time and to give underestimates when
the calibration point is older than the estimated time. A
similar conclusion was obtained by Rodriguez-Trelles,
Tarrio, and Ayala (2002) in their computer simulation. Our
observations support this theoretical prediction. These
biases are caused mainly by the variances and covariances
of a and b in equation (1). Therefore, if we use the con-
catenated sequences of many genes, these variances and
covariances are reduced, and consequently the estima-
tion bias is reduced, as is clear from tables 1 and 3. For
this reason, we recommend that the CS method rather than
the IG method be used in time estimation.
For estimating divergence times for distantly related
organisms, it has been customary to use protein sequences
rather than DNA sequences, because DNA sequences are
usually less conserved than protein sequences and the
substitution pattern in DNA sequences varies extensively
with codon position (Nei and Kumar 2000, chapters 2 and
3). In the present study, PC gamma distance for nuclear
proteins gave reasonably good time estimates for simian
primates, and the gamma parameter value (a) did not affect
the results seriously as long as a was greater than the
estimated one. In the case of DNA sequences the estimates
depended on the codon positions and the a value used. In
general, Kimura distance with a 5 for first and second
codon position data gave reasonable estimates, but Kimura
gamma distance with the estimated a value for all three
codon positions or first and second codon positions did
not. Therefore, it appears that protein sequences give more
robust estimates than DNA sequences, even for relatively
closely related species as long as many genes are used.
Divergence Times of Major Primate Lineages 431
However, this problem should be studied in more detail
considering both distantly related and closely related
species.
Many authors have used mt genes for time estima-
tion. The present study shows that even for relatively
closely related species such as simian primates these genes
do not give good results because the evolutionary rate
varies extensively among different evolutionary lineages.
It is therefore preferable to use nuclear genes rather than
mitochondrial genes for time estimation.
As the genome sequencing project proceeds in various
organisms, we will have many genes that can be used for
time estimation. However, because the number of gene
sequences available usually varies from species to species,
the number of genes shared by all species is often rather
small. For this reason, some authors (e.g., Stauffer et al.
2001) have used only three species or three groups of species
at a time for time estimation without using outgroups. This
approach certainly increases the number of genes available,
but the molecular clock cannot be tested properly unless the
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root of the three-species tree is determined by using
outgroups (Takezaki, Rzhetsky, and Nei 1995). Therefore,
this method may not give accurate estimates even if a large
number of genes is used. The different sets of genes used for
different triplet of species or species groups may also give
different time estimates. It is generally advisable first to
construct a phylogenetic tree for all the species involved and
then estimate divergence times.
In the present article we used two calibration dates for
estimating divergence times to see whether the two different
dates give similar estimates or not. This investigation was
useful in identifying the right kind of molecular data and the
right kind of statistical methods. However, once we know
the best data set or the best statistical method, we can now
use the two or more calibration dates to obtain a more
reliable evolutionary rate by using the regression method,
as was done by Hughes and Nei (1990) and Takahashi,
Rooney, and Nei (2000). If the calibration dates are reliable,
this method would give more reliable time estimates. In
practice, however, there are cases in which one calibra-
tion point is more reliable than others (Kumar and Hedges
1998). If this is the case, use of one or two reliable calibration
points may be preferable.
A number of authors (e.g., Sanderson 1997; Rambaut
and Bromham 1998; Kishino, Thorne, and Bruno 2001;
Soltis et al. 2002) have used sophisticated statistical
methods to take care of rate variation in the presence of
multiple calibration dates. However, even these methods
do not seem to work well when the extent of rate variation
is large (Soltis et al. 2002). It appears that the most
important thing for obtaining reliable time estimates is to
use molecular data that follow the molecular clock more
closely than others.
Some authors estimated divergence times by using
several local evolutionary rates in different parts of the tree
(e.g., Yoder and Yang 2000). If we know local evolution-
ary rate very accurately, this approach is expected to give
reliable time estimates. In practice, however, the local
(relative) rates are determined intuitively by looking at the
branch lengths of the original phylogenetic tree. Therefore,
the results obtained are often different depending on
432 Glazko and Nei
the number of local rates and the calibration points used.
For example, Yoder and Yang (2000) estimated the human/
chimpanzee divergence time using mitochondrial genes
from 31 mammalian species. They used different sub-
stitution models, different data types (protein sequences,
and different codon positions of DNA sequences), different
numbers of local clocks, and different calibration points
and obtained various estimates ranging from 2.68 to 10.12
MYA. It was therefore difficult to choose the most likely
estimate from the statistical analysis alone, and they chose
estimates that were most likely to agree with the available
fossil record. We also analyzed our mt gene data using the
Yoder and Yang method with two or four local clocks, but
the results were no better than those obtained by the stem-
lineage method shown in table 5 (see online Supplementary
Material). This again suggests that mt gene data are not
appropriate for time estimation in primates, whatever
method is used. What is important in time estimation is
to use genes that follow the global molecular clock as much
as possible.
Note that the estimation of divergence times from
molecular data is not to fit molecular data to the fossil
record available. Fossil records are usually very poor in
providing divergence time estimates as mentioned below,
and the utility of molecular clocks is to provide time
estimates that are difficult to obtain from the fossil record.
Therefore, a global clock that applies at least to a group of
species is necessary.
In this article, we used several different data sets and
distance measures each with a single global clock. We
obtained relatively close but slightly different time
estimates using different distance measures. For example,
our estimate of the time of divergence between humans
and chimpanzees obtained by the CS method varied from
5.5 MYA to 7.4 MYA in table 1 and table 3 (excluding the
standard errors), the average of the 12 observations being
6.3 MYA. (Although the estimates in table 3 are based on
a subset of the genes used in table 1, we treated them as
independent estimates because the estimates depend on the
genes and distance measures used and we wanted to know
only a crude magnitude of variation of the estimates
without consideration of standard errors.) Therefore, we
can probably say that the divergence between humans and
chimpanzees occurred about 6 MYA with a rough range of
57 MYA. If we use this crude approach, the times of
divergence of the human lineage from the gorilla,
orangutan, OW monkey, and NW monkey lineages
become 7 MYA (range, 68), 13 MYA (range, 1215),
23 MYA (range, 2125), and 33 MYA (range, 3236).
Here we included the fossil estimate (13 MYA) for the
computation of the human/orangutan divergence and used
only the estimates in table 3 for the computation of
the human/OW monkey and the human/NW monkey
divergence.
Note that the above estimates were obtained without
consideration of uncertainty of fossil dating. We used T1 5
90 MYA for the primate/artiodactyl divergence, but the
actual dating of Ungulatomorpha varies from 85 MYA to
90 MYA. The dating of Sivapithecus also varies from
6.8 MYA to 12.7 MYA (Ward 1997). Furthermore, these
dates do not necessarily indicate the actual time of species
divergence (Easteal 1999). Therefore, the actual time of
divergence may deviate even more from our estimates. In
the presence of this uncertainty, what kind of estimates
should we trust? In our opinion, the best way would be to
construct linearized trees for a group of species (many
different species of primates in the present case) using
several different groups of genes and examine the
consistency among time estimates obtained from different
sets of genes. If different genes give similar estimates, we
can accept them until they are rejected by other new sets of
genes.
If we know the uncertainty of calibration points, it is
clear that the standard error computed here is only a small
portion of the uncertainty of time estimates. The
magnitude of a standard error also depends on the method
of computation. In this study, we computed the standard
error of a time estimate from concatenated sequences using
genes as the units of bootstrap resampling. Theoretically, it
is possible to compute this standard error using amino
acids (or nucleotides) as the units of resampling. However,
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the latter method gives an unduly small standard error,
because the unit of evolution is a gene rather than an
amino acid. In the IG method the magnitude of standard
error is determined in part by the extent of elimination of
outliers as mentioned earlier. Because the extent of
elimination of outliers is subjective, the reliability of
standard errors is difficult to evaluate. For these reasons,
the standard error attached to a time estimate does not give
a real extent of uncertainty, and we should not place much
emphasis on it.
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